PROJECT SUMMARY High prevalence of HIV-associated neurocognitive disorders (HAND) poses a major challenge for the society. While there is evidence that both HIV-1 and methamphetamine can cause behavioral, neurocognitive and histopathological changes in the brain, and affect immune defense, the combined effect and potential interaction of both the virus and the drug remains incompletely understood. In addition, fundamental and critical questions regarding how HIV- infects microglia to establish latent infection and infected microglia alter neural tissue structure, physiology, and function in brain remain to be addressed. The overall objective of this proposal is to delineate the molecular and cellular mechanisms by which HIV infection alters the function of microglia, resident immune cells of the brain, and CNS that leads to neuronal injury and pathogenesis of HAND. Recent advances in embryonic stem cell and induced pluripotent stem cells (iPSCs) technologies have opened up new avenues of disease modeling in vitro. We put forward an unprecedented concept and experimental system to model human microglia-brain interactions as well as opportunities to illuminate how genetic variation affects gene expression. Our project has three specific aims: Specific Aim 1: Establish iPSC-derived cerebral organoids and define the cellular diversity, neural tissue structure, physiology, and gene expression programs. Specific Aim 2: Determine how HIV infection alters microglia and CNS during HIV neuropathogenesis. Specific Aim 3: Determine the molecular mechanisms of neuronal injury by HIV. Our results will be corroborated with gene expression programs using single nuclei of microglia and neurons obtained from postmortem tissues of HAND patients. Gene targets of HAND risk variants activity in disease-relevant cell types will be determined. Further, we will validate our findings in human brain tissues through the National NeuroAIDS Tissue Consortium (NNTC) and SIV infected brains of rhesus monkeys. Results of these studies will provide fundamental understanding of the molecular mechanisms that are altered by HIV and methamphetamine use leading to immune and brain dysfunctions.